1. Field of the Invention
The present invention relates to a fluorescence detection method, and a fluorescence detection apparatus useful for immunoassay. Particularly the present invention relates to a fluorescence detection method and an apparatus for determining the concentration of an objective substance with high accuracy by measuring the fluorescence from the objective substance and the fluorescence from a reference substance.
2. Description of the Related Art
Methods of immunoassay are known which utilize a fluorescent label contained in a immunocomplex or a fluorescent product formed by a labeling substance. Of these methods, enzyme-labeled immunoassay is conducted, for example, by bringing into contact a serum sample containing an objective substance and an antibody labeled by an enzyme with an antibody immobilized on a solid phase to form an immunocomplex, removing the unreacted portion of the enzyme-labeled antibody, adding a substrate for the enzyme contained in the complex to cause an enzyme reaction, and measuring the fluorescence emitted from the fluorescent substance formed by the enzyme reaction.
The conventional fluorescence detection apparatus for such a detection method comprises an exciting light irradiation system having a light source and a lens, and an optical measurement system comprising at least one detection element and at least one lens for measuring the fluorescence emitted from an objective substance (fluorescent substance). The optical system is selected to have the direction of the projected exciting light (illuminating light) so as to coincide substantially with, or to be perpendicular to the converging direction of the emitted fluorescence, depending on the measurement cell type and the surrounding conditions. In the optical system having the exciting light direction coinciding with the fluorescent light emission direction, a partial reflection mirror such as a dichroic mirror is employed to separate the fluorescent light from the exciting light.
The conventional detection apparatuses have disadvantages such that the measurement accuracy is not sufficient owing to variation of the measured value caused by change of the meniscus of the sample surface or by the presence of bubbles, and that the linear dependence of the fluorescence intensity on the concentration of the objective substance is impaired, at high concentrations, owing to absorption of the exciting light by the objective substance, causing the drop of the intensity of the generated fluorescence.
To offset such disadvantages, a method is proposed (JP-A-5-38297 and its corresponding U.S. Pat. No. 5,460,943). In this method, a reference fluorescent substance is added which is excited at the same wavelength as the objective substance in the sample and emits fluorescence of a wavelength different from that of the objective measurement substance, and the fluorescent light intensities of the sample and the reference substance are measured to obtain the concentration of the objective substance. This method, which utilizes different fluorescence wavelengths, may be called a two-wavelength fluorometry. For example, in this method, the fluorescence light is separated into two fluorescence light beams by a partial reflection mirror such as a dichroic mirror, the separated fluorescence light beams are respectively allowed to pass through a wavelength selection element, thus the fluorescence from the objective substance and the fluorescence from the reference substance are detected selectively, the intensities of the respective fluorescence light are measured, and the measured value of the fluorescence light of the objective substance is corrected based on the change of the fluorescence light of the reference substance.
The two-wavelength fluorometry enables high accuracy measurement. The inventors of the present invention made studies to improve the accuracy further, and found that the ratio of the changes of the signals of the two fluorescence intensities are not always constant in measurement of many samples in the aforementioned conventional two-wavelength fluorometry.
The variation of the ratio of the signal changes was found to be due to the causes below. For example, in the apparatus for immunoassay for quantitative determination of a biological substance, enzyme-containing complexes are placed usually in relatively small cells, and a large number of such cells are delivered mechanically and automatically into an optical measurement station for the quantitative determination. However, it is not easy to deliver constantly the cells without positional variation precisely to the predetermined position in the measurement station. In particular, in commercial apparatuses utilizing a large number of measurement cells, it is extremely difficult to reproducibly position each cell with respect to the optical measurement system. The positional variation of the cells occurs unavoidably. Furthermore, bubbles can be formed in the measurement liquid in dispensing the enzyme substrate into the measurement cell, and the bubble formation cannot be completely prevented. The variation of the signal change ratio of the aforementioned two detection elements, by this positional deviation and the bubble formation makes impossible the complete correction for the positional deviation. When the cell deviates positionally or bubbles are formed in the cell, the ratio of the signal changes of the two detectors is not constant in the two-wavelength fluorometry owing to physical non-equivalency such as positional deviations of the detection elements, of the apertures before the respective detection elements, and of the optical axes of the respective detection elements, and sensitivity irregularity of the detection elements, thereby causing inconformity of the change ratios of the detection signals with the two detection elements.
In the case where the intensity of the fluorescence from the reference substance is affected more by the positional deviation of the cell than that from the measurement object substance, the measurement data corrected by the same procedure (by use of a correction equation, or the like) involves the influence of the positional deviation.